Deep venous thrombosis (DVT) and pulmonary embolism (PE) remain major, common problems in clinical medicine. They result in significant morbidity and mortality and expensive health care costs. This project will develop a Two-Step procedure in experimental animals for the diagnosis of both DVT and PE by planar gamma camera imaging. The extraordinarily high affinity of streptavidin (SA) for biotin will be utilized in developing this Two-Step imaging technology. A galactose modified targeting agent is being developed that will exhibit high binding affinity and specificity to blood clots with the unbound agent clearing from the circulation by the galactose receptors in the liver. Utilizing genetic engineering, we are producing the single chain Fv (scFv) fragment of GC4. The scFv will then be fused by genetic engineering with the SA gene to produce a recombinant scFv-SA fusion monomer. The monomers self anneal to form the native SA structure consisting of 4 scFvs and 4 SA biotin binding sites. The scFv-SA targeting agent will exhibit high avidity to thrombi and emboli due to its multivalent antigen binding sites. The recombinant scFv-SA will then be galactose-modified using our isothiocyanate-trigalacose derivative (ITC-Tgal) successfully developed during the phase I portion of this grant. The scFv-SA conjugates will be characterized in vitro for immunoreactivity and biotin binding and in vivo for thrombus/embolus imaging in experimental animals. Furthermore, we are developing a new biotin chelate for Tc-99m labeling. The physical characteristics of Tc- 99m and the rapid blood clearance of radiolabeled biotin make it an ideal derivative for clinical imaging. PROPOSED COMMERCIAL APPLICATION The commercial application of the research (and subsequent product development effort) will be to provide a single noninvasive diagnostic procedure for both deep venous thrombosis (DVT) and pulmonary embolism (PE). In the U.S. an estimated 2.5 million cases of DVT and 600,00 cases of PE occur each year leading to 100,000 deaths and contributing to another 100,000. Due to the inadequacies of current diagnostic methods, multiple tests are often needed, some of which are expensive and entail risks to the patient.